Human microfibril-associated glycoprotein 4 splice variant

ABSTRACT

The invention provides a human microfibril-associated glycoprotein 4 splice variant (MAG4V) and polynucleotides which identify and encode MAG4V. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of MAG4V.

This application is a divisional application of U.S. application Ser.No. 09/008,960, filed Jan. 20, 1998.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of ahuman microfibril-associated glycoprotein 4 splice variant and to theuse of these sequences in the diagnosis, treatment, and prevention ofdevelopmental, reproductive, muscle, immunological, and neoplasticdisorders.

BACKGROUND OF THE INVENTION

Many eukaryotic cells are enveloped by an extracellular matrix ofproteins that provide structural support, cell and tissue identity, andautocrine, paracrine and juxtacrine properties for the cell within itsenvironment. (McGowan, S. E. (1992) FASEB J. 6:2895-2904.) The diversebiochemistry of extracellular matrix (ECM) proteins is indicative of themany, often overlapping, roles that are attributed to each distinctmolecule. (Grant, D. S. and Kleinman, H. K. (1997) EXS 79:317-333.)Whilst a great number of ECM proteins have been isolated, it remainsunclear how the majority of ECM proteins interact with one another orwith other molecules residing within the cell membrane.

Many ECM proteins have been associated with tissue growth and cellproliferation, others with tissue or cell differentiation, and yetothers with cell death. (Taipale, J. and Keski-Oja, J. (1997) FASEB J.11:51-59; and Eleftheriou, C. S. et al. (1991) Mutat. Res. 256:127-138.)For example, the process of embryonic bone formation involves thecreation of an extracellular matrix that mineralizes during the courseof tissue maturation. During the life of an individual, this matrix issubject to constant remodeling through the combined actions ofosteoblasts, which form mineralized bone, and osteoclasts, which resorbbone. The balance of ECM composition, and the resulting bone structure,may be perturbed by biochemical changes that result from congenital,epigenetic, or infectious diseases. (Francomano, C. A. et al. (1996)Curr. Opin. Genet. Dev. 6:301-308.)

ECM proteins also act as important mediators and regulators during theinflammatory response. Leukocytes are primed for inflammatory mediatorand cytokine production by binding to ECM proteins during extravasation.(Pakianathan, D. R. (1995) J. Leukoc. Biol. 57:699-702 ) Deposition ofECM proteins is also triggered by inflammation in response to lunginjury. Although the function of newly deposited matrices in injuredlungs is unknown. their ability to affect the migration, proliferation,differentiation, and activation state of cells in vitro suggests animportant role in the initiation and maintenance of the inflammatoryresponse in vivo. (Roman. J. (1996) Immunol. Res. 15:163-178.)

Multidomain or mosaic proteins play an important role in the diversefunctions of the ECM. ECM proteins are frequently characterized by thepresence of one or more domains which may include collagen-like domains,EGF-like domains, immunoglobulin-like domains, fibronectin-like domains,and von Willebrand Factor A-like modules. (Ayad. S. et al. (1994) TheExtracellular Matrix FactsBook, Academic Press. San Diego, Calif. pp4-7.)

Cell adhesion molecules are located in the plasma membrane and associatewith the ECM. They have been shown to stimulate axonal growth throughhomophilic and/or heterophilic interactions with other molecules. Forexample, proteins that contain the Arg-Gly-Asp (RGD) attachment site,and the integrins that serve as their receptors, constitute a majorrecognition system for cell adhesion. In addition, interactions betweenadhesion molecules and their receptors can potentiate the effects ofgrowth factors upon cell biochemistry via shared signaling pathways.(Ruoslahti, E. (1997) Kidney Int. 51:1413-1417.)

Integrins are ubiquitous transmembrane adhesion molecules that linkcells to the ECM by interacting with the cytoskeleton. Integrins alsofunction as signal transduction receptors and stimulate changes inintracellular calcium levels and protein kinase activity. (Sjaastad, M.D. and Nelson. W. J. (1997) BioEssays 19:47-55.) For example,fibronectin is recognized by at least ten cell surface receptors of theintegrin family which mediate the involvement of fibronectin in manydifferent biological processes.

The composition of the ECM is also regulated by differential proteolyticactivity. Cysteine proteases (e.g., cathepsin) are produced bymonocytes, macrophages and other immune cells and are involved indiverse cellular processes ranging from the processing of precursorproteins to intracellular degradation. Overproduction of these enzymescan cause the tissue destruction associated with rheumatoid arthritisand asthma.

Smith-Magenis syndrome (SMS) is a multiple congenital anomaly and mentalretardation syndrome associated with the deletion of human chromosome17p11.1. In normal tissue, chromosome 17p11.1 contains a gene encodingmicrofibril-associated glycoprotein 4 (MFAP4) in addition to severalother genes which are homologous to known eukaryotic proteins. (Chen, K.S. et al. (1995) Am. J. Hum. Genet. 56:175-182; and Koyama, K. et al.(1996) Cytogenet. Cell. Genet. 72:78-82.) The MFAP4 gene is deleted inalmost all SMS patients studied. MFAP4 has a fibrinogen-like domain andthe N-terminus has an RGD sequence which suggests that the gene encodesan ECM protein involved in cell adhesion or intracellular interactions.(Zhao, Z. et al. (1995) Hum. Mol. Genet. 4:589-597.) Furthermore. thegene encoding MFAP2, a candidate gene for involvement in the etiology ofinherited connective tissue diseases, contains two alternatively used 5'untranslated exons. (Faraco, J. et al. (1995) Genomics 25:630-637.)

The discovery of a new human microfibril-associated glycoprotein 4splice variant and the polynucleotides encoding it satisfies a need inthe art by providing new compositions which are useful in the diagnosis,treatment, and prevention of developmental, reproductive. muscle,immunological, and neoplastic disorders.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, humanmicrofibril-associated glycoprotein 4 splice variant (MAG4V), consistingof the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention further provides a substantially purified fragment ofMAG4V consisting of residues 1 through 27 of the amino acid sequence ofSEQ ID NO:1. The invention also provides an isolated and purifiedpolynucleotide encoding the polypeptide consisting of the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention alsoincludes an isolated and purified polynucleotide variant having at least90% polynucleotide identity to the polynucleotide encoding thepolypeptide consisting of the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1.

Additionally, the invention provides a composition comprising apolynucleotide encoding the polypeptide consisting of the amino acidsequence ot SEQ ID NO:1 or a fragment of SEQ ID NO:1. The inventionfurther provides an isolated and purified polynucleotide whichhybridizes under stringent conditions to the polynucleotide encoding thepolypeptide consisting of the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1, as well as an isolated and purifiedpolynucleotide which is complementary to the polynucleotide encoding thepolypeptide consisting of the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1.

The invention also provides an isolated and purified polynucleotidecomprising a sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, andan isolated and purified polynucleotide variant having at least 90%polynucleotide identity to the polynucleotide comprising the sequence ofSEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides anisolated and purified polynucleotide which is complementary to thepolynucleotide comprising the sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2. The invention also provides a polynucleotide fragmentcomprising nucleotides 1-135 of SEQ ID NO:2.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide consisting ofthe amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. Inanother aspect, the expression vector is contained within a host cell.

The invention also provides a method for producing a polypeptideconsisting of the amino acid sequence of SEQ ID NO:1 or a fragment ofSEQ ID NO:1, the method comprising the steps of: (a) culturing the hostcell containing an expression vector containing at least a fragment of apolynucleotide encoding MAG4V under conditions suitable for theexpression of the polypeptide; and (b) recovering the polypeptide fromthe host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified MAG4V having the sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1 in conjunction with a suitable pharmaceuticalcarrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, as well as a purified agonist and a purified antagonist of thepolypeptide.

The invention also provides a method for treating or preventing adevelopmental disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified MAG4V.

The invention also provides a method for treating or preventing areproductive disorder, the method comprising administering to a subjectin need of such treatment an effective amount of an antagonist of MAG4V.

The invention also provides a method for treating or preventing a muscledisorder, the method comprising administering to a subject in need ofsuch treatment an effective amount of an antagonist of MAG4V.

The invention also provides a method for treating or preventing animmunological disorder, the method comprising administering to a subjectin need of such treatment an effective amount of an antagonist of MAG4V.

The invention also provides a method for treating or preventing aneoplastic disorder, the method comprising administering to a subject inneed of such treatment an effective amount of an antagonist of MAG4V.

The invention also provides a method for detecting a polynucleotideencoding MAG4V in a biological sample containing nucleic acids, themethod comprising the steps of:

(a) hybridizing the complement of the polynucleotide encoding thepolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1 to at least one of the nucleic acids of the biological sample,thereby forming a hybridization complex; and (b) detecting thehybridization complex, wherein the presence of the hybridization complexcorrelates with the presence of a polynucleotide encoding MAG4V in thebiological sample. In one aspect, the nucleic acids of the biologicalsample are amplified by the polymerase chain reaction prior to thehybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, and 1E show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of MAG4V. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.Ltd. San Bruno. Calif.).

FIGS. 2A and 2B show the amino acid sequence alignments between MAG4V(Incyte Clone number: SEQ ID NO:1), and human MFAP4 (GI 790817: SEQ IDNO:3), produced using the multisequence alignment program of DNASTARsoftware (DNASTAR Inc. Madison Wis.).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a," "an," and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to "ahost cell" includes a plurality of such host cells, and a reference to"an antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Definitions

"MAG4V," as used herein, refers to the amino acid sequences ofsubstantially purified MAG4V obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term "agonist," as used herein, refers to a molecule which, whenbound to MAG4V, increases or prolongs the duration of the effect ofMAG4V. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of MAG4V.

An "allele" or an "allelic sequence," as these terms are used herein, isan alternative form of the gene encoding MAG4V. Alleles may result fromat least one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toalleles are generally ascribed to natural deletions, additions, orsubstitutions of nucleotides. Each of these types of changes may occuralone, or in combination with the others, one or more times in a givensequence.

"Altered" nucleic acid sequences encoding MAG4V, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same MAG4V or apolypeptide with at least one functional characteristic of MAG4V.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding MAG4V, and improper or unexpected hybridizationto alleles, with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding MAG4V. The encoded protein may also be"altered," and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent MAG4V. Deliberate amino acid substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of MAG4Vis retained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine.

The terms "amino acid" or "amino acid sequence," as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, "fragments", "immunogenic fragments", or"antigenic fragments" refer to fragments of MAG4V which are preferablyabout 5 to about 15 amino acids in length and which retain somebiological activity or immunological activity of MAG4V. Where "aminoacid sequence" is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, "amino acid sequence" and liketerms are not meant to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

"Amplification," as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,pp. 1-5.)

The term "antagonist," as it is used herein, refers to a molecule which,when bound to MAG4V, decreases the amount or the duration of the effectof the biological or immunological activity of MAG4V. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of MAG4V.

As used herein, the term "antibody" refers to intact molecules as wellas to fragments thereof, such as Fab F(ab')₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindMAG4V polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term "antigenic determinant," as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e. the immunogen used to elicit the immune response) for binding toan antibody.

The term "antisense," as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to a specificnucleic acid sequence. The term "antisense strand" is used in referenceto a nucleic acid strand that is complementary to the "sense" strand.Antisense molecules may be produced by any method including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes and to block either transcription or translation. Thedesignation "negative" can refer to the antisense strand, and thedesignation "positive" can refer to the sense strand.

As used herein, the term "biologically active," refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic MAG4V, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity," as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A." Complementaritybetween two single-stranded molecules may be "partial," such that onlysome of the nucleic acids bind, or it may be "complete," such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

A "composition comprising a given polynucleotide sequence" or a"composition comprising a given amino acid sequence," as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding MAG4V orfragments of MAG4V may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS), and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

The phrase "consensus sequence," as used herein, refers to a nucleicacid sequence which has been resequenced to resolve uncalled bases,extended using XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/orthe 3' direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW fragment assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

As used herein, the term "correlates with expression of apolynucleotide" indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding MAG4V, bynorthern analysis is indicative of the presence of nucleic acidsencoding MAG4V in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding MAG4V.

A "deletion," as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term "derivative," as used herein, refers to the chemicalmodification of MAG4V, of a polynucleotide sequence encoding MAG4V, orof a polynucleotide sequence complementary to a polynucleotide sequenceencoding MAG4V. Chemical modifications of a polynucleotide sequence caninclude, for example, replacement of hydrogen by an alkyl, acyl, oramino group. A derivative polynucleotide encodes a polypeptide whichretains at least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

The term "homology," as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology. Theword "identity" may substitute for the word "homology." A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of reduced stringency. This is not tosay that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e. a selective)interaction. The absence of non-specific binding may be tested by theuse of a second target sequence which lacks even a partial degree ofcomplementarity (e.g., less than about 30% homology or identity). In theabsence of non-specific binding, the substantially homologous sequenceor probe will not hybridize to the second non-complementary targetsequence.

The phrases "percent identity" or "% identity" refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (Lasergene softwarepackage, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can createalignments between two or more sequences according to different methods,e.g., the clustal method. (Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no homology between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be calculated by theclustal method, or by other methods known in the art, such as the JotunHein method. (See, e.g., Hein, J. (1990) Methods in Enzymology183:626-645.) Identity between sequences can also be determined by othermethods known in the art, e.g., by varying hybridization conditions.

"Human artificial chromosomes" (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat. Genet. 15:345-355.)

The term "humanized antibody," as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

"Hybridization," as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term "hybridization complex" as used herein, refersto a complex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary bases. A hybridizationcomplex may be formed in solution (e.g., C₀ t or R₀ t analysis) orformed between one nucleic acid sequence present in solution and anothernucleic acid sequence immobilized on a solid support (e.g., paper,membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

The words "insertion" or "addition," as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

"Immune response" can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term "microarray," as used herein, refers to an array of distinctpolynucleotides or oligonucleotides arrayed on a substrate, such aspaper, nylon or any other type of membrane, filter, chip, glass slide,or any other suitable solid support.

The term "modulate," as it appears herein, refers to a change in theactivity of MAG4V. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of MAG4V.

The phrases "nucleic acid" or "nucleic acid sequence," as used herein,refer to an oligonucleotide, nucleotide, polynucleotide, or any fragmentthereof, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In this context, "fragments" refers to those nucleicacid sequences which are greater than about 60 nucleotides in length,and most preferably are at least about 100 nucleotides, at least about1000 nucleotides, or at least about 10,000 nucleotides in length.

The terms "operably associated" or "operably linked," as used herein,refer to functionally related nucleic acid sequences. A promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the transcription of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in reading frame, certain genetic elements, e.g.,repressor genes, are not contiguously linked to the encoded polypeptidebut still bind to operator sequences that control expression of thepolypeptide.

The term "oligonucleotide." as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term"oligonucleotide" is substantially equivalent to the terms "amplimers,""primers," "oligomers," and "probes," as these terms are commonlydefined in the art.

"Peptide nucleic acid" (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA and RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

The term "sample," as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding MAG4V.or fragments thereof, or MAG4V itself may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

As used herein, the terms "specific binding" or "specifically binding"refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein recognized by thebinding molecule (i.e., the antigenic determinant or epitope). Forexample, if an antibody is specific for epitope "A," the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term "stringent conditions" refers to conditionswhich permit hybridization between polynucleotide sequences and theclaimed polynucleotide sequences. Suitably stringent conditions can bedefined by, for example, the concentrations of salt or formamide in theprehybridization and hybridization solutions, or by the hybridizationtemperature, and are well known in the art. In particular, stringencycan be increased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5× SSPE, 0.3%SDS, and 200 μg/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term "substantially purified," as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A "substitution," as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation," as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term "transformed" cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, and refers to cells which transiently express the insertedDNA or RNA for limited periods of time.

A "variant" of MAG4V, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have "nonconservative" changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The Invention

The invention is based on the discovery of a new humanmicrofibril-associated glycoprotein 4 splice variant (MAG4V), thepolynucleotides encoding MAG4V, and the use of these compositions forthe diagnosis, treatment, or prevention of developmental, reproductive,muscle, immunological, and neoplastic disorders.

Nucleic acids encoding the MAG4V of the present invention were firstidentified in Incyte Clone 1361119 from the lung cDNA library(LUNGNOT12) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2. was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones1361119 (LUNGNOT12), 639925 (BRSTNOT03), 2209783 (SINTFET03), 0687195(UTRSNOT02), 1515436 (PANCTUT01), 1659231 (URETTUT01), and 1300012(BRSTNOT07).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1, as shown in FIGS. 1A, 1B, 1C,1D, and 1E. MAG4V is 279 amino acids in length and has two potentialN-glycosylation sites at residues N-111 and N-161; two potential caseinkinase II phosphorylation sites at residues S-179 and S-120; threepotential protein kinase C phosphorylation sites at residues T-16,T-145, and S-268; an RGD cell attachment sequence from R-50 to D-52; afibrinogen-like domain from about P-62 to about R-277; and a potentialsignal peptide sequence from M-1 to about S-40. As shown in FIGS. 2A and2B, MAG4V has chemical and structural homology with human MFAP4 (GI709817; SEQ ID NO:3). In particular, MAG4V and human MFAP4 share 91%identity, the two potential N-glycosylation sites, the two potentialcasein kinase II phosphorylation sites, two potential protein kinase Cphosphorylation sites, the RGD site, and the fibrinogen-like domain. Aregion from amino acid residues 1 through 27 of SEQ ID NO: 1, which ispresent only in MAG4V, may be used as an immunogenic or antigenicpolypeptide. The fragment of SEQ ID NO:2 from about nucleotide 1 toabout nucleotide 135 is useful for designing oligonucleotides or for useas a hybridization probe. Northern analysis shows the expression of thissequence in various libraries, at least 54% of which are immortalized orcancerous and at least 18% of which involve immune response. Ofparticular note is the expression of MAG4V in gastrointestinal,developmental, fetal, muscle, and connective tissues, and in lung,heart, prostate, uterus, breast, bladder, and penis tissues.

The invention also encompasses MAG4V variants. A preferred MAG4V variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe MAG4V amino acid sequence, and which contains at least onefunctional or structural characteristic of MAG4V.

The invention also encompasses polynucleotides which encode MAG4V. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2. which encodes an MAG4V.

The invention also encompasses a variant of a polynucleotide sequenceencoding MAG4V. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding MAG4V. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2 which has at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2. Any one ofthe polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of MAG4V.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding MAG4V, some bearing minimal homology to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring MAG4V, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode MAG4V and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring MAG4V under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding MAG4V or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding MAG4V and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences which encodeMAG4V and MAG4V derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding MAG4V or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, or a fragment of SEQ ID NO:2,under various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407, and Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.)

Methods for DNA sequencing are well known and generally available in theart and may be used to practice any of the embodiments of the invention.The methods may employ such enzymes as the Klenow fragment of DNApolymerase I, SEQUENASE (US Biochemical Corp., Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(GIBCO/BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the MICROLAB 2200 (Hamilton, Reno, Nev.), Peltierthermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABICATALYST and 373 and 377 DNA SEQUENCERS (Perkin Elmer).

The nucleic acid sequences encoding MAG4V may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus. (See, e.g., Sarkar, G. (1993) PCRMethods Applic. 2:318-322.) In particular, genomic DNA is firstamplified in the presence of a primer complementary to a linker sequencewithin the vector and a primer specific to the region predicted toencode the gene. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region. (See, e.g., Triglia, T. etat. (1988) Nucleic Acids Res. 16:8186.) The primers may be designedusing commercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc. Plymouth, Minn.) or anotherappropriate program to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to 72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR, which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to place anengineered double-stranded sequence into an unknown fragment of the DNAmolecule before performing PCR. Other methods which may be used toretrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060.) Additionally, one mayuse PCR, nested primers, and PROMOTERFINDER™ libraries to walk genomicDNA (Clontech, Palo Alto, Calif.). This process avoids the need toscreen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will include moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5' non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separations four differentfluorescent dyes (one for each nucleotide) which are laser activated,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g. GENOTYPER and SEQUENCENAVIGATOR, Perkin Elmer), and the entire process from loading of samplesto computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode MAG4V may be used in recombinant DNAmolecules to direct expression of MAG4V. or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressMAG4V.

As will be understood by those of skill in the art, it may beadvantageous to produce MAG4V-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alterMAG4V-encoding sequences for a variety of reasons including, but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding MAG4V may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of MAG4V activity, it may be useful toencode a chimeric MAG4V protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the MAG4V encoding sequence and theheterologous protein sequence, so that MAG4V may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding MAG4V may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, the protein itself may be produced usingchemical methods to synthesize the amino acid sequence of MAG4V, or afragment thereof. For example, peptide synthesis can be performed usingvarious solid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995)Science 269:202-204.) Automated synthesis may be achieved using the ABI431A peptide synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or by sequencing. (See, e.g., Creighton, T. (1983) Proteins,Structures and Molecular Properties, W H Freeman and Co., New York,N.Y.) Additionally, the amino acid sequence of MAG4V, or any partthereof, may be altered during direct synthesis and/or combined withsequences from other proteins, or any part thereof, to produce a variantpolypeptide.

In order to express a biologically active MAG4V, the nucleotidesequences encoding MAG4V or derivatives thereof may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding MAG4V andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding MAG4V. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The "control elements" or "regulatory sequences" are thosenon-translated regions, e.g., enhancers, promoters, and 5' and 3'untranslated regions, of the vector and polynucleotide sequencesencoding MAG4V which interact with host cellular proteins to carry outtranscription and translation. Such elements may vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. For example, whencloning in bacterial systems, inducible promoters, e.g., hybrid lacZpromoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) orPSPORT1 plasmid (GIBCO/BRL), may be used. The baculovirus polyhedrinpromoter may be used in insect cells. Promoters or enhancers derivedfrom the genomes of plant cells (e.g., heat shock, RUBISCO, and storageprotein genes) or from plant viruses (e.g., viral promoters or leadersequences) may be cloned into the vector. In mammalian cell systems,promoters from mammalian genes or from mammalian viruses are preferable.If it is necessary to generate a cell line that contains multiple copiesof the sequence encoding MAG4V, vectors based on SV40 or EBV may be usedwith an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for MAG4V. For example, when largequantities of MAG4V are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to,multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding MAG4V may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced, and pIN vectors. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) pGEX vectors(Pharmacia Biotech, Uppsala, Sweden) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters, such as alpha factor, alcoholoxidase, and PGH, may be used. (See, e.g., Ausubel, supra; and Grant etal. (1987) Methods Enzymol. 153:516-544.)

In cases where plant expression vectors are used, the expression ofsequences encoding MAG4V may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;Broglie, R. et al. (1984) Science 224:838-843; and Winter. J. et al.(1991) Results Probl. Cell Differ. 17:85-105.) These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews. (See, e.g., Hobbs, S. or Murry,L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGrawHill, New York, N.Y.; pp. 191-196.)

An insect system may also be used to express MAG4V. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding MAG4V may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of sequences encoding MAG4V will render the polyhedrin geneinactive and produce recombinant virus lacking coat protein. Therecombinant viruses may then be used to infect, for example, S.frugiperda cells or Trichoplusia larvae in which MAG4V may be expressed.(See, e.g., Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci.91:3224-3227.)

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding MAG4V may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing MAG4V in infected host cells. (See, e.g., Logan,J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) Inaddition, transcription enhancers, such as the Rous sarcoma virus (RSV)enhancer, may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding MAG4V. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding MAG4V and its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularcell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl.Cell Differ. 20:125-162.)

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding,and/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293. and W138), are available fromthe American Type Culture Collection (ATCC, Bethesda, Md.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

For long term, high yield production of recombinant proteins, stableexpression is preferred. For example, cell lines capable of stablyexpressing MAG4V can be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for about 1 to 2 days in enriched media before being switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase genes and adenine phosphoribosyltransferase genes,which can be employed in tk⁻ or apr⁻ cells, respectively. (See, e.g.,Wigler, M. et al. (1977) Cell 11:223-232; and Lowy, I. et al. (1980)Cell 22:817-823) Also, antimetabolite, antibiotic, or herbicideresistance can be used as the basis for selection. For example, dhfrconfers resistance to methotrexate, npt confers resistance to theaminoglycosides neomycin and G-418; and als and pat confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively. (See,e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570;Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14; and Murry,supra.) Additional selectable genes have been described, e.g., trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine. (See,e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.85:8047-8051.) Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, β glucuronidase and itssubstrate GUS, luciferase and its substrate luciferin. Green fluorescentproteins (GFP) (Clontech. Palo Alto, Calif.) are also used (See, e.g.,Chalfie, M. et al. (1994) Science 263:802-805.) These markers can beused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system. (See, e.g., Rhodes, C. A. et al. 1995) Methods Mol. Biol.55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingMAG4V is inserted within a marker gene sequence, transformed cellscontaining sequences encoding MAG4V can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding MAG4V under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding MAG4V and express MAG4V may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA--DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

The presence of polynucleotide sequences encoding MAG4V can be detectedby DNA--DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding MAG4V. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding MAG4V to detect transformantscontaining DNA or RNA encoding MAG4V.

A variety of protocols for detecting and measuring the expression ofMAG4V, using either polyclonal or monoclonal antibodies specific for theprotein, are known in the art. Examples of such techniques includeenzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on MAG4V is preferred, but a competitivebinding assay may be employed. These and other assays are well describedin the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; and Maddox, D.E. et al. (1983) J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding MAG4V includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding MAG4V,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by Pharmacia &Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. BiochemicalCorp. (Cleveland, Ohio). Suitable reporter molecules or labels which maybe used for ease of detection include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding MAG4V may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeMAG4V may be designed to contain signal sequences which direct secretionof MAG4V through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding MAG4V to nucleotidesequences encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAG extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences, such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.), between the purificationdomain and the MAG4V encoding sequence may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing MAG4V and a nucleic acid encoding 6 histidineresidues preceding: a thioredoxin or an enterokinase cleavage site. Thehistidine residues facilitate purification on immobilized metal ionaffinity chromatography. (IMIAC) (See, e.g., Porath, J. et al. (1992)Prot. Exp. Purif. 3:263-281.) The enterokinase cleavage site provides ameans for purifying MAG4V from the fusion protein. (See, e.g., Kroll, D.J. et al. (1993) DNA Cell Biol. 12:441-453.)

Fragments of MAG4V may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, T. E. (1984) Protein: Structures and MolecularProperties, pp. 55-60, W. H. Freeman and Co. New York, N.Y.) Proteinsynthesis may be performed by manual techniques or by automation.Automated synthesis may be achieved, for example, using the AppliedBiosystems 431A peptide synthesizer (Perkin Elmer). Various fragments ofMAG4V may be synthesized separately and then combined to produce thefull length molecule.

Therapeutics

Chemical and structural homology exists between MAG4V and human MFAP4(GI 790817). In addition, MAG4V is expressed in cancer and proliferatingtissues; in cells of the immune response; in gastrointestinal,developmental, fetal, muscle, and connective tissues, and in lung,heart, prostate, uterus, breast, bladder, and penis tissues. Therefore,MAG4V appears to play a role in developmental, reproductive, muscle,immunological, and neoplastic disorders.

Therefore, in one embodiment, MAG4V or a fragment or derivative thereofmay be administered to a subject to treat or prevent a developmentaldisorder. The term "developmental disorder" refers to any disorderassociated with development or function of a tissue, organ, or system ofa subject (such as the brain, adrenal gland, kidney, skeletal orreproductive system). Such developmental disorders can include, but arenot limited to, renal tubular acidosis, anemia, Cushing's syndrome,achondroplastic dwarfism, Duchenne and Becker muscular dystrophy,epilepsy, gonadal dysgenesis, WAGR syndrome, Smith-Magenis syndrome,myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spinal bifida, congenital glaucoma, cataract, and sensorineuralhearing loss.

In another embodiment, a vector capable of expressing MAG4V or afragment or derivative thereof may be administered to a subject to treator prevent a developmental disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified MAG4V in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a developmental disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofMAG4V may be administered to a subject to treat or prevent adevelopmental disorder including, but not limited to, those listedabove.

In a further embodiment, an antagonist of MAG4V may be administered to asubject to treat or prevent a reproductive disorder. Such a reproductivedisorder may include, but is not limited to, disorders of prolactinproduction; infertility, including tubal disease, ovulatory defects, andendometriosis; disruptions of the estrous cycle, disruptions of themenstrual cycle, polycystic ovary syndrome, ovarian hyperstimulationsyndrome, endometrial and ovarian tumors, autoimmune disorders, ectopicpregnancy, and teratogenesis; cancer of the breast, fibrocystic breastdisease, and galactorrhea; disruptions of spermatogenesis, abnormalsperm physiology, cancer of the testis, cancer of the prostate, benignprostatic hyperplasia, and prostatitis, carcinoma of the male breast andgynecomastia. In one aspect, an antibody which specifically binds MAG4Vmay be used directly as an antagonist or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to cells ortissue which express MAG4V.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding MAG4V may be administered to a subject to treator prevent a reproductive disorder including, but not limited to, thosedescribed above.

In a further embodiment, an antagonist of MAG4V may be administered to asubject to treat or prevent a muscle disorder. Such a muscle disordermay include, but is not limited to, cardiomyopathy, myocarditis,Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonicdystrophy, central core disease, nemaline myopathy, centronuclearmyopathy, lipid myopathy, mitochondrial myopathy, infectious myositis,polymyositis, dermatomyositis, inclusion body myositis, thyrotoxicmyopathy, and ethanol myopathy. In one aspect, an antibody whichspecifically binds MAG4V may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express MAG4V.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding MAG4V may be administered to a subject to treator prevent a muscle disorder including, but not limited to, thosedescribed above.

In a further embodiment, an antagonist of MAG4V may be administered to asubject to treat or prevent an immunological disorder. Such animmunological disorder may include, but is not limited to, AIDS,Addison's disease, adult respiratory distress syndrome, allergies,ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, erythema nodosum,atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout,Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, ulcerative colitis, Werner syndrome,and complications of cancer, hemodialysis, and extracorporealcirculation; viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections; and trauma. In one aspect, an antibody whichspecifically binds MAG4V may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express MAG4V.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding MAG4V may be administered to a subject to treator prevent an immunological disorder including, but not limited to,those described above.

In a further embodiment, an antagonist of MAG4V may be administered to asubject to treat or prevent a neoplastic disorder. Such a neoplasticdisorder may include, but is not limited to, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus. In one aspect, an antibody which specifically binds MAG4V may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress MAG4V.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding MAG4V may be administered to a subject to treator prevent a neoplastic disorder including, but not limited to, thosedescribed above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of MAG4V may be produced using methods which are generallyknown in the art. In particular, purified MAG4V may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind MAG4V. Antibodies to MAG4V may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith MAG4V or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to MAG4V have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of MAG4V aminoacids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to MAG4V may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of "chimericantibodies," such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce MAG4V-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi. R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-3837, and Winter. G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for MAG4V mayalso be generated. For example, such fragments include, but are notlimited to, F(ab')2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab')2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between MAG4V and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering MAG4V epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

In another embodiment of the invention, the polynucleotides encodingMAG4V, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect. the complement of thepolynucleotide encoding MAG4V may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding MAG4V. Thus, complementary molecules orfragments may be used to modulate MAG4V activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding MAG4V.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencescomplementary to the polynucleotides of the gene encoding MAG4V. (See,e.g., Sambrook, supra; and Ausubel, supra.)

Genes encoding MAG4V can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide,or fragment thereof, encoding MAG4V. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5', or regulatory regions of the gene encodingMAG4V. Oligonucleotides derived from the transcription initiation site,e.g., between about positions -10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingMAG4V.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding MAG4V. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the molecule,or the use of phosphorothioate or 2'O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl- , methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of MAG4V,antibodies to MAG4V, and mimetics, agonists, antagonists, or inhibitorsof MAG4V. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution. Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of MAG4V. such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example MAG4V or fragments thereof, antibodies of MAG4V,and agonists, antagonists or inhibitors of MAG4V, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED50 (the dosetherapeutically effective in 50% of the population) or LD50 (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD50/ED50 ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind MAG4V may beused for the diagnosis of disorders characterized by expression ofMAG4V, or in assays to monitor patients being treated with MAG4V oragonists, antagonists, or inhibitors of MAG4V. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for MAG4V include methodswhich utilize the antibody and a label to detect MAG4V in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

A variety of protocols for measuring MAG4V, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of MAG4V expression. Normal or standard values for MAG4Vexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toMAG4V under conditions suitable for complex formation The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of MAG4V expressed insubject, samples from biopsied tissues are compared with the standardvalues. Deviation between standard and subject values establishes theparameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingMAG4V may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofMAG4V may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of MAG4V, and tomonitor regulation of MAG4V levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding MAG4V or closely related molecules may be used to identifynucleic acid sequences which encode MAG4V. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding MAG4V, alleles,or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe MAG4V encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the MAG4V gene.

Means for producing specific hybridization probes for DNAs encodingMAG4V include the cloning of polynucleotide sequences encoding MAG4V orMAG4V derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³² p or ³⁵ S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding MAG4V may be used for the diagnosis ofa disorder associated with expression of MAG4V. Examples of such adisorder include, but are not limited to, a developmental disorder suchas, renal tubular acidosis, anemia, Cushing's syndrome, achondroplasticdwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadaldysgenesis, WAGR syndrome, Smith-Magenis syndrome, myelodysplasticsyndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas,hereditary neuropathies such as Charcot-Marie-Tooth disease andneurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders suchas Syndenham's chorea and cerebral palsy, spinal bifida, congenitalglaucoma, cataract, and sensorineural hearing loss; a reproductivedisorder such as, disorders of prolactin production; infertility,including tubal disease, ovulatory defects, and endometriosis;disruptions of the estrous cycle, disruptions of the menstrual cycle,polycystic ovary syndrome, ovarian hyperstimulation syndrome,endometrial and ovarian tumors, autoimmune disorders, ectopic pregnancy,and teratogenesis; cancer of the breast, fibrocystic breast disease, andgalactorrhea; disruptions of spermatogenesis, abnormal sperm physiology,cancer of the testis, cancer of the prostate, benign prostatichyperplasia, and prostatitis, carcinoma of the male breast andgynecomastia; a muscle disorder such as, cardiomyopathy, myocarditis,Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonicdystrophy, central core disease, nemaline myopathy, centronuclearmyopathy, lipid myopathy, mitochondrial myopathy, infectious myositis,polymyositis, dermatomyositis, inclusion body myositis, thyrotoxicmyopathy, and ethanol myopathy; an immunological disorder such as, AIDS,Addison's disease, adult respiratory distress syndrome, allergies,ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, erythema nodosum,atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout,Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, ulcerative colitis, Werner syndrome,and complications of cancer, hemodialysis, and extracorporealcirculation; viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections; and trauma, and a neoplastic disorder such as,adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. The polynucleotidesequences encoding MAG4V may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies: indipstick, pin, and ELISA assays; and in microarrays utilizing fluids ortissues from patients to detect altered MAG4V expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding MAG4V may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingMAG4V may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding MAG4V in the sample indicatesthe presence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of MAG4V, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding MAG4V, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding MAG4V may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding MAG4V, or a fragment of a polynucleotide complementary to thepolynucleotide encoding MAG4V, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of MAG4Vinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby. P. C. et al. (1993) J. Immunol. Methods 159:235-244;and Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

In one embodiment, the microarray is prepared and used according tomethods known in the art. (See, e.g., Chee et al. (1995) PCT applicationWO95/11995; Lockhart. D. J. et al. (1996) Nat. Biotech. 14:1675-1680;and Schena. M. et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619.)

The microarray is preferably composed of a large number of uniquesingle-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs. The oligonucleotidesare preferably about 6 to 60 nucleotides in length, more preferablyabout 15 to 30 nucleotides in length, and most preferably about 20 to 25nucleotides in length. It may be preferable to use oligonucleotideswhich are about 7 to 10 nucleotides in length. The microarray maycontain oligonucleotides which cover the known 5' or 3' sequence,sequential oligonucleotides which cover the full length sequence, orunique oligonucleotides selected from particular areas along the lengthof the sequence. Polynucleotides used in the microarray may beoligonucleotides specific to a gene or genes of interest.Oligonucleotides can also be specific to one or more unidentified cDNAsassociated with a particular cell type or tissue type. It may beappropriate to use pairs of oligonucleotides on a microarray. The firstoligonucleotide in each pair differs from the second oligonucleotide byone nucleotide. This nucleotide is preferably located in the center ofthe sequence. The second oligonucleotide serves as a control. The numberof oligonucleotide pairs may range from about 2 to 1,000,000.

In order to produce oligonucleotides for use on a microarray, the geneof interest is examined using a computer algorithm Which starts at the5' end, or, more preferably, at the 3' end of the nucleotide sequence.The algorithm identifies oligomers of defined length that are unique tothe gene, have a GC content within a range suitable for hybridization,and lack secondary structure that may interfere with hybridization. Inone aspect, the oligomers may be synthesized on a substrate using alight-directed chemical process. (See, e.g., Chee et al., supra.) Thesubstrate may be any suitable solid support, e.g., paper, nylon, anyother type of membrane, or a filter, chip, or glass slide.

In another aspect, the oligonucleotides may be synthesized on thesurface of the substrate using a chemical coupling procedure and an inkjet application apparatus. (See, e.g., Baldeschweiler et al. (1995) PCTapplication WO95/251116.) An array analogous to a dot or slot blot(HYBRIDOT apparatus. GIBCO/BRL) may be used to arrange and link cDNAfragments or oligonucleotides to the surface of a substrate using avacuum system or thermal, UV, mechanical, or chemical bondingprocedures. An array may also be produced by hand or by using availabledevices, materials, and machines, e.g. BRINKMANN multichannel pipettorsor robotic instruments. The array may contain from 2 to 1,000,000 or anyother feasible number of oligonucleotides.

In order to conduct sample analysis using the microarrays,polynucleotides are extracted from a sample. The sample may be obtainedfrom any bodily fluid, e.g., blood, urine, saliva, phlegm, gastricjuices, cultured cells, biopsies, or other tissue preparations. Toproduce probes, the polynucleotides extracted from the sample are usedto produce nucleic acid sequences complementary to the nucleic acids onthe microarray. If the microarray contains cDNAs, antisense RNAs (aRNAs)are appropriate probes. Therefore, in one aspect, mRNA isreverse-transcribed to cDNA. The cDNA, in the presence of fluorescentlabel, is used to produce fragment or oligonucleotide aRNA probes. Thefluorescently labeled probes are incubated with the microarray so thatthe probes hybridize to the microarray oligonucleotides. Nucleic acidsequences used as probes can include polynucleotides, fragments, andcomplementary or antisense sequences produced using restriction enzymes,PCR, or other methods known in the art.

Hybridization conditions can be adjusted so that hybridization occurswith varying degrees of complementarity. A scanner can be used todetermine the levels and patterns of fluorescence after removal of anynonhybridized probes. The degree of complementarity and the relativeabundance of each oligonucleotide sequence on the microarray can beassessed through analysis of the scanned images. A detection system maybe used to measure the absence, presence, or level of hybridization forany of the sequences. (See, e.g., Heller, R. A. et al. (1997) Proc.Natl. Acad. Sci. 94:2150-2155.)

In another embodiment of the invention, nucleic acid sequences encodingMAG4V may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, VCH Publishers New York. N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding MAG4V on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., AT to 11q22-23. any sequences mapping to that area may representassociated or regulatory genes for further investigation. (See, e.g.,Gatti, R. A. et al. (1988) Nature 336:577-580.) The nucleotide sequenceof the subject invention may also be used to detect differences in thechromosomal location due to translocation, inversion, etc., amongnormal, carrier, or affected individuals.

In another embodiment of the invention, MAG4V, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenMAG4V and the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with MAG4V, orfragments thereof, and washed. Bound MAG4V is then detected by methodswell known in the art. Purified MAG4V can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding MAG4V specificallycompete with a test compound for binding MAG4V. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with MAG4V.

In additional embodiments, the nucleotide sequences which encode MAG4Vmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. LUNGNOT12 cDNA Library Construction

The LUNGNOT12 cDNA library was constructed from microscopically normallung tissue obtained from a 78-year-old Caucasian male who had undergonea segmental lung resection following diagnosis of malignant neoplasm ofthe right upper lobe. The pathology report indicated invasive pulmonarygrade 3 adenocarcinoma forming a peripheral mass with associatedfibrosis. The fibrosis pleura was puckered, but not invaded.Additionally, the patient exhibited ventricular premature beats andchronic airway obstruction due to extrinsic asthma. The pathology reportalso indicated a history of cerebrovascular disease, arterioscleroticvascular disease, thrombophlebitis, malignant neoplastic prostate, andprevious tobacco abuse which was in remission. The patient familyhistory included cerebrovascular disease, arteriosclerotic vasculardisease, and Type I diabetes in patient's siblings.

The frozen tissue was homogenized and lysed using a Polytron PT-3000Homogenizer (Brinkmann Instruments, Westbury, N.J.) in guanidiniumisothiocyanate solution. The lysate was centrifuged over a 5.7 M CsClcushion using a Beckman SW28 rotor in a Beckman L8-70 M Ultracentrifuge(Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature.The RNA was extracted with acid phenol pH 4.7, precipitated using 0.3 Msodium acetate and 2.5 volumes of ethanol, resuspended in RNase-freewater, and treated with DNase at 37° C. The RNA was extracted andprecipitated as before. The mRNA was then isolated using the OLIGOTEXkit (QIAGEN, Inc., Chatsworth, Calif.) and used to construct the cDNAlibrary.

The mRNA was handled according to the recommended protocols in theSUPERSCRIPT plasmid system (Catalog #18248-013, GIBCO-BRL). cDNAsynthesis was initiated with a NotI-oligo d(T) primer. Double-strandedcDNA was blunted, ligated to EcoRI adaptors, digested with NotI,fractionated on a SEPHAROSE CL4B column (Catalog #275105-01, Pharmacia),and those cDNAs exceeding 400 bp were ligated into the NotI and EcoRIsites of the plasmid PSPORT1 (Catalog #15382-013, GIBCO-BRL). Theplasmid PSPORT1 was subsequently transformed into DH5α competent cells(Catalog #18258-012, GIBCO-BRL).

II. Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the R.E.A.L.PREP 96 plasmid kit (Catalog #26173, QIAGEN, Inc.). The recommendedprotocol was employed except for the following changes:1) the bacteriawere cultured in 1 ml of sterile Terrific Broth (Catalog #22711,GIBCO-BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%, 2) afterinoculation, the cultures were incubated for 19 hours and at the end ofincubation, the cells were lysed with 0.3 ml of lysis buffer; and 3)following isopropanol precipitation, the plasmid DNA pellet wasresuspended in 0.1 ml of distilled water. After the last step in theprotocol, samples were transferred to a 96-well block for storage at 4°C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441), using a MICROLAB 2200 (Hamilton, Reno, Nev.) incombination with Peltier thermal cyclers (PTC200 from MJ Research,Watertown, Mass.) and 377 DNA sequencing systems and the reading framewas determined.

III. Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; and Altschul et al.(1990) J. Mol. Biol. 215:403-410.)

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms couldhave been used when dealing with primary sequence patterns and secondarystructure gap penalties. (See, e.g., Smith, T. et al. (1992) ProteinEngineering 5:35-51.) The sequences disclosed in this application havelengths of at least 49 nucleotides and have no more than 12% uncalledbases (where N is recorded rather than A, C, G, or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdseas set at 10⁻²⁵ for nucleotides and 10⁻⁸ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam),and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for homology.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; andAusubel, F. M. et al. supra, ch. 4 and 16.) Analogous computertechniques applying BLAST are used to search for identical or relatedmolecules in nucleotide databases such as GenBank or LIFESEQ database(Incyte Pharmaceuticals). This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score, which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Homologous molecules are usually identified by selecting thosewhich show product scores between 15 and 40, although lower scores mayidentify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding MAG4V occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of MAG4V Encoding Polynucleotides

The nucleic acid sequence of Incyte Clone 1361119 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension of anantisense polynucleotide, and the other was synthesized to initiateextension of a sense polynucleotide. Primers were used to facilitate theextension of the known sequence "outward" generating ampliconscontaining new unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06software (National Biosciences, Plymouth, Minn.), or another appropriateprogram, to be about 22 to 30 nucleotides in length, to have a GCcontent of about 50% or more, and to anneal to the target sequence attemperatures of about 68° C. to about 72° C. Any stretch of nucleotideswhich would result in hairpin structures and primer--primerdimerizations was avoided.

Selected human cDNA libraries (GIBCO/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the Peltier thermal cyclers(PTC200; M.J. Research, Watertown, Mass.), beginning with 40 pmol ofeach primer and the recommended concentrations of all other componentsof the kit, with the following parameters:

    ______________________________________                                        Step 1    94° C. for 1 min (initial denaturation)                                 Step 2 65° C. for 1 min                                       Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat steps 4 through 6 for an additional 15 cycles                   Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat steps 8 through 10 for an additional 12 cycles                 Step 12 72° C. for 8 min                                               Step 13  4° C. (and holding)                                         ______________________________________                                    

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQuick (QIAGEN Inc., Chatsworth, Calif.),and trimmed of overhangs using Klenow enzyme to facilitate religationand cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A. p. 1) containing 2× Carb. Thefollowing day, several colonies were randomly picked from each plate andcultured in 150 μl of liquid LB/2× Carb medium placed in an individualwell of an appropriate commercially-available sterile 96-well microtiterplate. The following day, 5 μl of each overnight culture was transferredinto a non-sterile 96-well plate and, after dilution 1:10 with water, 5μl from each sample was transferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1    94° C. for 60 sec                                              Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2 through 4 for an additional 29 cycles                   Step 6 72° C. for 180 sec                                              Step 7  4° C. (and holding)                                          ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5' regulatory sequences using the procedure above, oligonucleotidesdesigned for 5' extension, and an appropriate genomic library.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³² P] adenosinetriphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide kinase(DuPont NEN, Boston, Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine resin column(Pharmacia & Upjohn, Kalamazoo. Mich.). An aliquot containing 10⁷ countsper minute of the labeled probe is used in a typical membrane-basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II(DuPont NEN, Boston, Mass.).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots, hybridization patternsare compared visually.

VII. Microarrays

To produce oligonucleotides for a microarray, one of the nucleotidesequences of the present invention is examined using a computeralgorithm which starts at the 3' end of the nucleotide sequence. Foreach, the algorithm identifies oligomers of defined length that areunique to the nucleic acid sequence, have a GC content within a rangesuitable for hybridization, and lack secondary structure that wouldinterfere with hybridization. The algorithm identifies approximately 20oligonucleotides corresponding to each nucleic acid sequence. For eachsequence-specific oligonucleotide, a pair of oligonucleotides issynthesized in which the first oligonucleotides differs from the secondoligonucleotide by one nucleotide in the center of the sequence. Theoligonucleotide pairs can be arranged on a substrate, e.g. a siliconchip, using a light-directed chemical process. (See, e.g., Chee, supra.)

In the alternative, a chemical coupling procedure and an ink jet devicecan be used to synthesize oligomers on the surface of a substrate. (See,e.g., Baldeschweiler, supra.) An array analogous to a dot or slot blotmay also be used to arrange and link fragments or oligonucleotides tothe surface of a substrate using or thermal, UV, mechanical, or chemicalbonding procedures, or a vacuum system. A typical array may be producedby hand or using available methods and machines and contain anyappropriate number of elements. After hybridization, nonhybridizedprobes are removed and a scanner used to determine the levels andpatterns of fluorescence. The degree of complementarity and the relativeabundance of each oligonucleotide sequence on the microarray may beassessed through analysis of the scanned images.

VIII. Complementary Polynucleotides

Sequences complementary to the MAG4V-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring MAG4V. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software andthe coding sequence of MAG4V. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5' sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the MAG4V-encoding transcript.

IX. Expression of MAG4V

Expression of MAG4V is accomplished by subcloning the cDNA into anappropriate vector and transforming the vector into host cells. Thisvector contains an appropriate promoter, e.g., β-galactosidase upstreamof the cloning site, operably associated with the cDNA of interest.(See, e.g., Sambrook, supra, pp. 404-433; and Rosenberg, M. et al.(1983) Methods Enzymol. 101:123-138.)

Induction of an isolated, transformed bacterial strain with isopropylbeta-D-thiogalactopyranoside (IPTG) using standard methods produces afusion protein which consists of the first 8 residues ofβ-galactosidase, about 5 to 15 residues of linker, and the full lengthprotein. The signal residues direct the secretion of MAG4V intobacterial growth media which can be used directly in the following assayfor activity.

X. Demonstration of MAG4V Activity

The activity of human microfibril-associated glycoprotein 4 splicevariant may be measured using an assay based upon the property of ECMproteins to support in vitro proliferation of fibroblasts and tumorcells under serum-free conditions. (Chiquet-Ehrismann, R. et al. (1986)Cell 47:131-139.) Wells in 96 well cluster plates (Falcon, FisherScientific, Santa Clara, Calif.) are coated with MAG4V by incubationwith solutions at 50-100 μg MAG4V/ml for 15 min at ambient temperature.The coating solution is aspirated, and the wells washed with Dulbecco'smedium before cells are plated. Rat fibroblast cultures or rat mammarytumor cells are prepared as described and plated at a density of 10⁴-10⁵ cells/ml in Dulbecco's medium supplemented with 10% fetal calfserum. (Chiquet-Ehrismann, R. et al. supra.)

After three days the medium is removed, and the cells washed three timeswith phosphate-buffered saline (PBS), pH 7.0, before addition ofserum-free Dulbecco's medium containing 0.25 mg/ml bovine serum albumin(BSA. Fraction V, Sigma Chemical Company, St. Louis, Mo.). After 2 daysthe medium is aspirated, and 100 μl of [3H]thymidine (NEN) at 2 μCi/mlin fresh Dulbecco's medium containing 0.25 mg/ml BSA is added. Parallelplates are fixed and stained to determine cell numbers. After 16 hr, themedium is aspirated, the cell layer washed with PBS, and the 10%trichloroacetic acid-precipitable radioactivity in the cell layerdetermined by liquid scintillation counting, and normalized to relativecell numbers. (Chiquet-Ehrismann, R. et al. supra.)

XI. Production of MAG4V Specific Antibodies

MAG4V substantially purified using PAGE electrophoresis (see, e.g.,Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or otherpurification techniques, is used to immunize rabbits and to produceantibodies using standard protocols. The MAG4V amino acid sequence isanalyzed using DNASTAR software (DNASTAR Inc) to determine regions ofhigh immunogenicity, and a corresponding oligopeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Methods for selection of appropriate epitopes, such as those near theC-terminus or in hydrophilic regions are well described in the art.(See, e.g., Ausubel et al. supra, ch. 11.)

Typically, the oligopeptides are 15 residues in length, and aresynthesized using an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry and coupled to KLH (Sigma. St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel et al. supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide activity, for example, bybinding the peptide to plastic, blocking with 1% BSA, reacting withrabbit antisera, washing, and reacting with radio-iodinated goatanti-rabbit IgG.

XII. Purification of Naturally Occurring MAG4V Using Specific Antibodies

Naturally occurring or recombinant MAG4V is substantially purified byimmunoaffinity chromatography using antibodies specific for MAG4V. Animmunoaffinity column is constructed by covalently coupling anti-MAG4Vantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing MAG4V are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of MAG4V (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/MAG4V binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andMAG4V is collected.

XIII. Identification of Molecules Which Interact with MAG4V

MAG4V, or biologically active fragments thereof, are labeled with ¹²⁵ IBolton-Hunter reagent, (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled MAG4V, washed, and anywells with labeled MAG4V complex are assayed. Data obtained usingdifferent concentrations of MAG4V are used to calculate values for thenumber, affinity, and association of MAG4V with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 3                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 279 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: LUNGNOT12                                                        (B) CLONE: 1361119                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Met Gly Glu Leu Ser Pro Leu Gln Arg Pro - #Leu Ala Thr Glu Gly        Thr                                                                               1               5 - #                 10 - #                 15             - -  Met Lys Ala Gln Gly Val Leu Leu Lys Leu - #Ala Leu Leu Ala Leu Pro                   20     - #             25     - #             30                  - -  Leu Leu Leu Leu Leu Ser Thr Pro Pro Cys - #Ala Pro Gln Val Ser Gly               35         - #         40         - #         45                      - -  Ile Arg Gly Asp Ala Leu Glu Arg Phe Cys - #Leu Gln Gln Pro Leu Asp           50             - #     55             - #     60                          - -  Cys Asp Asp Ile Tyr Ala Gln Gly Tyr Gln - #Ser Asp Gly Val Tyr Leu       65                 - # 70                 - # 75                 - # 80       - -  Ile Tyr Pro Ser Gly Pro Ser Val Pro Val - #Pro Val Phe Cys Asp Met                       85 - #                 90 - #                 95              - -  Thr Thr Glu Gly Gly Lys Trp Thr Val Phe - #Gln Lys Arg Phe Asn Gly                   100     - #            105     - #            110                 - -  Ser Val Ser Phe Phe Arg Gly Trp Asn Asp - #Tyr Lys Leu Gly Phe Gly               115         - #        120         - #        125                     - -  Arg Ala Asp Gly Glu Tyr Trp Leu Gly Leu - #Gln Asn Met His Leu Leu           130             - #    135             - #    140                         - -  Thr Leu Lys Gln Lys Tyr Glu Leu Arg Val - #Asp Leu Glu Asp Phe Glu       145                 - #150                 - #155                 -         #160                                                                             - -  Asn Asn Thr Ala Tyr Ala Lys Tyr Ala Asp - #Phe Ser Ile Ser Pro        Asn                                                                                              165 - #                170 - #                175            - -  Ala Val Ser Ala Glu Glu Asp Gly Tyr Thr - #Leu Phe Val Ala Gly Phe                   180     - #            185     - #            190                 - -  Glu Asp Gly Gly Ala Gly Asp Ser Leu Ser - #Tyr His Ser Gly Gln Lys               195         - #        200         - #        205                     - -  Phe Ser Thr Phe Asp Arg Asp Gln Asp Leu - #Phe Val Gln Asn Cys Ala           210             - #    215             - #    220                         - -  Ala Leu Ser Ser Gly Ala Phe Trp Phe Arg - #Ser Cys His Phe Ala Asn       225                 - #230                 - #235                 -         #240                                                                             - -  Leu Asn Gly Phe Tyr Leu Gly Gly Ser His - #Leu Ser Tyr Ala Asn        Gly                                                                                              245 - #                250 - #                255            - -  Ile Asn Trp Ala Gln Trp Lys Gly Phe Tyr - #Tyr Ser Leu Lys Arg Thr                   260     - #            265     - #            270                 - -  Glu Met Lys Ile Arg Arg Ala                                                      275                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1941 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: LUNGNOT12                                                        (B) CLONE: 1361119                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  CTCTGAGCAG AACTGACAGC ATGAAGGTAC GGGGCCCAGG GTCGGGGGAC - #TCATAGCAT    G    60                                                                         - -  GGGGAACTGA GCCCACTCCA GAGGCCCCTG GCCACAGAGG GCACTATGAA - #GGCACAAGG    A   120                                                                         - -  GTTCTCTTGA AACTCGCACT CCTGGCCCTG CCGCTGCTGC TGCTTCTCTC - #CACGCCCCC    G   180                                                                         - -  TGTGCCCCCC AGGTCTCCGG GATCCGAGGA GATGCTCTGG AGAGGTTTTG - #CCTTCAGCA    A   240                                                                         - -  CCCCTGGACT GTGACGACAT CTATGCCCAG GGCTACCAGT CAGACGGCGT - #GTACCTCAT    C   300                                                                         - -  TACCCCTCGG GCCCCAGTGT GCCTGTGCCC GTCTTCTGTG ACATGACCAC - #CGAGGGCGG    G   360                                                                         - -  AAGTGGACGG TTTTCCAGAA GAGATTCAAT GGCTCAGTAA GTTTCTTCCG - #CGGCTGGAA    T   420                                                                         - -  GACTACAAGC TGGGCTTCGG CCGTGCTGAT GGAGAGTACT GGCTGGGGCT - #GCAGAACAT    G   480                                                                         - -  CACCTCCTGA CACTGAAGCA GAAGTATGAG CTGCGAGTGG ACTTGGAGGA - #CTTTGAGAA    C   540                                                                         - -  AACACGGCCT ATGCCAAGTA CGCTGACTTC TCCATCTCCC CGAACGCGGT - #CAGCGCAGA    G   600                                                                         - -  GAGGATGGCT ACACCCTCTT TGTGGCAGGC TTTGAGGATG GCGGGGCAGG - #TGACTCCCT    G   660                                                                         - -  TCCTACCACA GTGGCCAGAA GTTCTCTACC TTCGACCGGG ACCAGGACCT - #CTTTGTGCA    G   720                                                                         - -  AACTGCGCAG CTCTCTCCTC AGGAGCCTTC TGGTTCCGCA GCTGCCACTT - #TGCCAACCT    C   780                                                                         - -  AATGGCTTCT ACCTAGGTGG CTCCCACCTC TCTTATGCCA ATGGCATCAA - #CTGGGCCCA    G   840                                                                         - -  TGGAAGGGCT TCTACTACTC CCTCAAACGC ACTGAGATGA AAATCCGCCG - #GGCCTGAAG    G   900                                                                         - -  GCTGGCCCCC TCAGGCACCT TTCCTCCCCT GGACACCCAT GGTCTCCATG - #AGTGCTCCC    T   960                                                                         - -  CTGCTGCCCC TGATGCATGC TTCTGCTGAT TCCCGAGCAC CAACTCCTTA - #CAAGGGGGC    C  1020                                                                         - -  TTGTGGCTCT CAGCCATGCC ACATCCCTGT CACACACCCA GGGCATCCAT - #TCCTAAGCC    A  1080                                                                         - -  GACCCGGCTC CCCTACACCT GAAGTTACAC TGCCAGCAGT TCCCCAGGCC - #TCTTCCGAG    A  1140                                                                         - -  GGCACATGGT TCTAGCCTGG ACCTGGCTGG GCTCCATGAG AATGAGTTGC - #CTCCACCCT    G  1200                                                                         - -  TCCCAACAGC TGACAGCCAG GAGCCACTCT CCCAGCTGCA GGCCTTTGTG - #GTCCATCTT    G  1260                                                                         - -  TCCTGCTTCC TCACTGTGGA CCCCTGTCTG GGCCACCCTA GTGTGCTAAG - #CTGAGCAGT    G  1320                                                                         - -  CAGTGTGAAC AGGGCCCATG GTGTATTCTA GGCCACAGCC CAGCACTCCT - #CTGGGCTGC    T  1380                                                                         - -  CTCAAACCAT GTCCCATCTT CAGCATCCCT CCCACCAACT TACTCCCCTG - #TGGTGAGTA    C  1440                                                                         - -  CGTGGAACCC CAGCCCACCT CACTATCATA CTCAGCTTCC CCTGATGGCC - #CATCCCAGC    C  1500                                                                         - -  CCTGAAGCTC TATGCCAAGA ACACAGCTAC CGCACACCAC CCTGAAACAG - #CCACAGCCA    A  1560                                                                         - -  GGTAGGCATG CATATGAGGT CTTCCCCATA CCCTCTGGGT GTTGAGAGGT - #TTAGCCACA    T  1620                                                                         - -  GAGGGAGCAG AGGACAATCT CTGCAGGGCT GGGAGTGGGT AGGGACTGAA - #GGTCTCAAT    A  1680                                                                         - -  AACCTTCAGA ACCTGAATGA ACTGGCTTCA TACACACAAA CATATTTGTT - #TATCCCCCA    A  1740                                                                         - -  ATGTAGGCAC CTGGCTCCTC CTTGCTCCCC TGCTGATGGT GTCCTACCCC - #GAACTCCAA    A  1800                                                                         - -  AATTACACCT GGAGTCAGGT GCAGAAGGGA ACCTTGTATT TCACAGGCCT - #CATTTTGAT    G  1860                                                                         - -  GCAAAAAGAC AGTGTAATAA TAACATAATA ATAATAAAAA TATAATACTG - #AAAAAAAAA    A  1920                                                                         - -  AAAAAAAAAA AAAAAAAAAA A          - #                  - #                    1941                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 256 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 790817                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Ser Met Lys Ala Leu Leu Ala Leu Pro Leu - #Leu Leu Leu Leu Ser Thr        1               5 - #                 10 - #                 15              - -  Pro Pro Cys Ala Pro Gln Val Ser Gly Ile - #Arg Gly Asp Ala Leu Glu                   20     - #             25     - #             30                  - -  Arg Phe Cys Leu Gln Gln Pro Leu Asp Cys - #Asp Asp Ile Tyr Ala Gln               35         - #         40         - #         45                      - -  Gly Tyr Gln Ser Asp Gly Val Tyr Leu Ile - #Tyr Pro Ser Gly Pro Ser           50             - #     55             - #     60                          - -  Val Pro Val Pro Val Phe Cys Asp Met Thr - #Thr Glu Gly Gly Lys Trp       65                 - # 70                 - # 75                 - # 80       - -  Thr Val Phe Gln Lys Arg Phe Asn Gly Ser - #Val Ser Phe Phe Arg Gly                       85 - #                 90 - #                 95              - -  Trp Asn Asp Tyr Lys Leu Gly Phe Gly Arg - #Ala Asp Gly Glu Tyr Trp                   100     - #            105     - #            110                 - -  Leu Gly Leu Gln Asn Met His Leu Leu Thr - #Leu Lys Gln Lys Tyr Glu               115         - #        120         - #        125                     - -  Leu Arg Val Asp Leu Glu Asp Phe Glu Asn - #Asn Thr Ala Tyr Ala Lys           130             - #    135             - #    140                         - -  Tyr Ala Asp Phe Ser Ile Ser Pro Asn Ala - #Val Ser Ala Glu Glu Asp       145                 - #150                 - #155                 -         #160                                                                             - -  Gly Tyr Thr Leu Phe Val Ala Gly Phe Glu - #Asp Gly Gly Ala Gly        Asp                                                                                              165 - #                170 - #                175            - -  Ser Leu Ser Tyr His Ser Gly Gln Lys Phe - #Ser Thr Phe Asp Arg Asp                   180     - #            185     - #            190                 - -  Gln Asp Leu Phe Val Gln Asn Cys Ala Ala - #Leu Ser Ser Gly Ala Phe               195         - #        200         - #        205                     - -  Trp Phe Arg Ser Cys His Phe Ala Asn Leu - #Asn Gly Phe Tyr Leu Gly           210             - #    215             - #    220                         - -  Gly Ser His Leu Ser Tyr Ala Asn Gly Ile - #Asn Trp Ala Gln Trp Lys       225                 - #230                 - #235                 -         #240                                                                             - -  Gly Phe Tyr Tyr Ser Leu Lys Arg Thr Glu - #Met Lys Ile Arg Arg        Ala                                                                                              245 - #                250 - #                255          __________________________________________________________________________

What is claimed is:
 1. A substantially purified humanmicrofibril-associated glycoprotein 4 splice variant (MAG4V) comprisingthe amino acid sequence of SEQ ID No:1 or amino acid residues 1 through27 of SEQ ID No:1.
 2. A substantially purified fragment of MAG4Vconsisting of amino acids 1 through 27 of the sequence of SEQ ID NO: 1.3. A composition comprising the MAG4V of claim 1.